Molecular marker related to waterlogging tolerance of cabbage type rape and application of molecular marker

Abstract

The invention belongs to the technical field of crop molecular markers, and particularly discloses a molecular marker related to the waterlogging tolerance of brassica napus and application of the molecular marker. According to the invention, a 1.2 Kb transposon insertion and a 2.6 Kb transposon insertion are identified by utilizing GWAS (Good White Attenual Sequence) and are positioned in a promoter region of the Bna.C08. GUN4 gene, and the rape containing the two transposon insertion has high waterlogging resistance. The molecular marker is used for auxiliary selection of oilseed rape with strong waterlogging resistance, whether the oilseed rape germplasm is waterlogging-resistant oilseed rape germplasm can be predicted only by detecting the characteristic band of the molecular marker, the identification method is simple and feasible, the selection efficiency is high, and the molecular marker has huge application potential in the field of oilseed rape waterlogging resistance improvement breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of crop molecular markers, and in particular relates to a molecular marker related to waterlogging tolerance of Brassica napus and its application. Background Art

[0002] Rapeseed is one of the most important oil crops in the world, accounting for about 50% of my country's vegetable oil supply. Rapeseed cultivation areas in my country are mainly concentrated in the Yangtze River Basin. Due to the widespread adoption of the "rice-oil" rotation cultivation model in the Yangtze River Basin, the soil in the paddy fields is heavy and the groundwater level is high after rice planting; coupled with the humid and rainy climate in the autumn and spring in the region, the risk of waterlogging in rapeseed production is relatively high. According to statistics, in the rapeseed planting areas in the Yangtze River Basin, the area of ​​waterlogging damage reaches 1.444 million hectares per year (mainly distributed in Sichuan, Chongqing, Hubei, Hunan, Anhui, Jiangsu and other places), accounting for more than 20% of the total area, resulting in a 17% to 42.4% reduction in rapeseed production. Waterlogging stress reduces the activity of photosynthesis-related enzymes, hinders chlorophyll synthesis and degrades, inhibits the formation of new leaves, and causes leaves to wilt, yellow, age and fall off, ultimately affecting the growth and development of the plants, and even leading to plant death; at the same time, waterlogging can cause a loss of grain yield in the mature stage of rapeseed, seriously affecting the production of rapeseed.

[0003] At present, the research on rapeseed's response to waterlogging mainly focuses on growth and physiological responses during stress, growth inhibition during maturity, etc. In recent years, comparative transcriptome and proteome analysis has been the main means of analyzing the genetic basis of rapeseed's response to waterlogging. With the widespread application of cutting-edge technologies such as genomics and phenotype groups in crop research, new opportunities have been brought about in the study of rapeseed's response to waterlogging. Genome-wide association study (GWAS) refers to a method of using molecular markers near genes or genes to perform association analysis with one or more complex traits at the genome-wide level to find genetic loci associated with target traits. It has now become a key means of identifying important agronomic traits.

[0004] Most of the previous studies focused on the physiological and morphological characteristics of rapeseed's response to waterlogging. Although the current research on plant waterlogging tolerance has progressed rapidly, most of them have stagnated at the stage of initial QTL positioning and excavation, while the research content of identifying functional genes and exploring their molecular mechanisms is relatively limited, and no waterlogging tolerance sites or key genes have been cloned so far. Therefore, in-depth research on rapeseed's response to waterlogging and its regulatory mechanism, discovering genetic loci and genes that regulate waterlogging response, and identifying germplasm with excellent waterlogging tolerance will help deepen the understanding of rapeseed's waterlogging response mechanism and provide a theoretical basis and germplasm resources for genetic improvement for breeding new varieties of waterlogging-tolerant rapeseed. The present invention uses GWAS based on structural variation (SV) to identify two adjacent transposon insertions of 1.2Kb and 2.6Kb in waterlogging-tolerant rapeseed germplasm, located in the promoter region of the Bna.C08.GUN4 gene. By designing primers for the TE insertion, a molecular marker closely linked to waterlogging tolerance is developed for molecular assisted selection of offspring with strong waterlogging tolerance in rapeseed breeding. Summary of the invention

[0005] The present invention determined the SPAD values ​​of 350 rapeseed germplasms planted at the Yangluo Experimental Base of the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences in Wuhan in 2023 after waterlogging treatment, and combined with the resequencing data, conducted a GWAS analysis based on structural variation, and identified a 1.2Kb and a 2.6Kb transposon insertion in the promoter region of the Bna.C08.GUN4 gene on chromosome C08 in the waterlogging-tolerant rapeseed. Thus, molecular markers related to waterlogging tolerance were developed for waterlogging tolerance identification and rapeseed breeding.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A molecular marker for identifying waterlogging tolerance of Brassica napus, comprising two 1.2Kb and 2.6Kb transposon insertions located in the promoter region of the Bna.C08.GUN4 gene, with sequences shown in SEQ ID NO.1 and SEQ ID NO.2.

[0008] A kit for identifying waterlogging tolerance of Brassica napus, the kit comprising reagents for detecting molecular markers of sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2. In a specific embodiment of the present invention, primers of sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4 are used to simultaneously detect the molecular markers.

[0009] A method for identifying waterlogging tolerance of Brassica napus, comprising detecting whether the rapeseed to be tested contains molecular markers of sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and the rapeseed containing the molecular markers has high waterlogging tolerance. In a specific embodiment of the present invention, the molecular markers are amplified using primers of sequences shown in SEQ ID NO.3 and 4, and if a product of 4.4 Kb is amplified, it has high waterlogging tolerance, and if a product of 537 bp is amplified, it has low waterlogging tolerance.

[0010] Compared with the prior art, the present invention can detect whether the target insert fragment is contained by conventional PCR and agarose gel, thereby judging whether the rapeseed variety is highly tolerant to waterlogging; this identification method is simple and feasible, and has high selection efficiency. Rapeseed varieties with strong waterlogging tolerance can be identified in the early growth stage of rapeseed, with a clear selection target and not affected by the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 (a) The frequency distribution diagram of SPAD values ​​of 350 materials in the control group and (b) the frequency distribution diagram of SPAD values ​​of 350 materials in the waterlogging group.

[0012] Figure 2 The results of SV-GWAS data analysis. a is the result of genome-wide association analysis of relative SPAD values ​​of 350 materials, and b is the location of the two identified transposons inserted on chromosome C08.

[0013] Figure 3 The relative SPAD value significance analysis of 350 associated groups. INS is a genotype containing 1.2 Kb and 2.6 Kb transposon insertions, and GUN4 is a genotype without 1.2 Kb and 2.6 Kb transposon insertions.

[0014] Figure 4 The genotype identification results of 5 waterlogging-tolerant and 5 waterlogging-intolerant rapeseeds (the marker used is 5K).

[0015] Figure 5 This is the growth trend of the control group and the waterlogging group after 7 days of waterlogging treatment.

[0016] Figure 6 Two genotypes (GUN4 and GUN4) before and after waterlogging treatment INS ) Relative expression level of BnaC08.GUN4 gene in materials.

[0017] Figure 7 The results of the two genotypes (GUN4 and GUN4) after 7 days of waterlogging treatment INS )Measurement results of physiological and biochemical indicators of materials.

[0018] Figure 8There are two genotypes (GUN4 and GUN4 INS )Comparison of promoter activities of materials. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with the examples. The test methods in the following examples are conventional test methods unless otherwise specified. The experimental reagents and consumables described in the following examples can be purchased from commercial companies unless otherwise specified. In this example, the detailed steps for screening molecular markers for rapeseed waterlogging tolerance materials are as follows:

[0020] 1. Rapeseed associated population planting and determination of SPAD value

[0021] 350 germplasm resource groups collected by the inventor's research group in the early stage were used as experimental materials. They were planted at the Yangluo Experimental Base of the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences in Wuhan in 2023 and were divided into a control group and a waterlogged experimental group. Each group was repeated three times, and each material was planted in double rows with a row length of 1.5m and a row spacing of 0.25m. The waterlogged treatment group began to be waterlogged when the rapeseed grew to the five-leaf stage one month after sowing. Field irrigation was used. The water level needed to be about 1-2cm above the soil surface, and waterlogging continued for ten days; the control group maintained conventional field management conditions during the growth period. After the waterlogged group was waterlogged for ten days, six plants with the same growth were selected from each material in the control group and the experimental group, and the SPAD value was determined using a SPAD instrument (SPAD-502Plus). The average value was calculated to obtain the final SPAD value of each material.

[0022] 2. Data Analysis

[0023] The relative value SPAD (SPAD value of waterlogging experimental group / SPAD value of control group) was used for SV-GWAS analysis. Combining 350 resequencing data, it was identified that there was a 1.2Kb (SEQ ID NO.1) and 2.6Kb (SEQ ID NO.2) transposon insertion on chromosome C08, located in the promoter region of the Bna.C08.GUN4 gene ( Figure 2 ), Bna.C08.GUN4 is a gene known to promote chlorophyll synthesis by activating magnesium chelatase activity. At the same time, a 700bp sequence insertion was identified on chromosome A10, located in the promoter region of the Bna.A10.DRM2 gene, but the Bna.A10.DRM2 gene has not yet been clearly functionally annotated, and compared with the C08 chromosome site, the SV-GWAS analysis results did not show a clustered increase, so no further analysis was performed.

[0024] 3.TE fragment amplification

[0025] The CTAB method was used to extract 350 genomic DNAs from the associated population, and the DNA quality was tested by 1% agarose gel electrophoresis. Primers were designed to amplify the two transposons inserted into the C08 chromosome, and the primer sequences are shown in SEQ ID NO.3 and 4. The PCR reaction system is shown in Table 1, the PCR amplification reaction procedure is shown in Table 2, and the amplified products were tested by 1% agarose gel electrophoresis. After all the tests were completed, the two genotype materials were distinguished and the relative SPAD value significance was calculated. Figure 3 As shown, GUN4 containing two transposon insertions INS The relative SPAD value of the genotype was significantly higher than that of the GUN4 genotype without transposon insertion.

[0026] Table 1 PCR reaction system

[0027] Components Volume (μL) 2x Phanta Flash Master Mix(Dye Plus) 10 Forward Primer 1 Reverse primer 1 <![CDATA[ddH2O]]> 7 DNA 1

[0028] Table 2 PCR amplification reaction program

[0029]

[0030] 4. Identification of two genotypes and phenotypes and determination of physiological and biochemical indicators

[0031] According to the data analysis results and the relative SPAD values, 5 materials containing two transposon insertions (8S006, 8S007, 8S091, 8S149, 8SA71) and 5 materials without transposon insertions (8S053, 8S190, 8S245, 8S292, 8SA59) were selected to measure physiological and biochemical indicators. The genotype identification results of the 10 materials are as follows: Figure 4 shown.

[0032] 4.1 Evaluation of indoor stain resistance of representative materials

[0033] Ten portions of material seeds were divided into 2mL centrifuge tubes and planted in 8L nutrient pots. Forty seeds were planted for each portion of material, and the solution used was Hoagland nutrient solution. On the first day of planting, dark culture was carried out first. After the radicle was exposed, the cover was opened and the light was turned on to maintain normal growth. The light: dark = 16:8h. After one week of growth, they were transplanted into 5×10 soil culture trays. Ten seedlings were transplanted for each portion of material. The soil used was nutrient soil purchased from the laboratory. After one week of normal growth, water was started when the seedlings grew to the two-leaf to three-leaf stage. The treatments were divided into a control group and a waterlogged group. The control group maintained conventional growth conditions during the growth process, and the waterlogged group maintained the water level 1-2cm above the soil surface during the growth process. Waterlogging was continued for 7 days. After 7 days, the growth conditions of the control group and the waterlogged group were as follows: Figure 5 As shown. Figure 5 It can be seen that there is no difference in the growth of the two genotypes in the control group, but after 7 days of waterlogging, GUN4INS Compared with the GUN4 genotype, the plant growth and leaf green retention of this genotype are stronger.

[0034] The young leaves of the control group and the treatment group of each material were selected to extract plant tissue RNA, and the RNA was reverse transcribed and then subjected to qRT-PCR experiments. The relative expression of the BnaC08.GUN4 gene before and after waterlogging was detected by comparing the waterlogged group with the control group. Figure 6 As shown, GUN4 INS After waterlogging, the expression of BnaC08.GUN4 gene in the GUN4 genotype increased compared with the control group, while the expression of BnaC08.GUN4 gene in the GUN4 genotype decreased. INS Among the materials with the same genotype, the relative expression level of BnaC08.GUN4 gene was significantly higher than that of GUN4 genotype.

[0035] Three plants with consistent growth were selected and the SPAD value was measured using a SPAD instrument (SPAD-502Plus); three materials with consistent growth were selected to weigh the fresh weight of the aboveground parts, three tissue samples of 0.2 g were weighed for the determination of malondialdehyde, and three tissue samples of 0.2 g were weighed for the determination of chlorophyll content.

[0036] like Figure 7 As shown in the figure, under the control condition (without waterlogging treatment), there were no significant differences in chlorophyll content, SPAD value, fresh weight of aboveground parts and MDA content between the two genotypes. However, under waterlogging treatment, GUN4 containing transposon insertion had a significant difference in chlorophyll content, SPAD value, fresh weight of aboveground parts and MDA content. INS The chlorophyll content, SPAD value and aboveground fresh weight of the genotype material were significantly higher than those of the GUN4 genotype material, and the MDA content was significantly lower than that of the GUN4 genotype material; this shows that the molecular markers developed by the present invention can be used to identify whether the rapeseed variety is a material with good waterlogging tolerance.

[0037] 4.2 Promoter activity of representative materials

[0038] In order to verify whether the insertion of TE has an effect on the promoter activity, we amplified the 2Kb fragments of the promoter region of the two genotypes and connected them to the pGreenII-0800-LUC vector. After transforming Escherichia coli DH5α, we performed single clone identification, and transferred the Escherichia coli-extracted plasmids of the positive clones into Agrobacterium GV3101. Then, the Agrobacterium tumefaciens strains (GV3101) carrying different plasmids were injected into the leaves of Nicotiana benthamiana. Within 48-72 hours, the luciferase reaction solution was evenly applied on the back of the tobacco, and the reaction was carried out in the dark for about 5 minutes. The LUC fluorescence was observed under a live imager. At least six independent leaves were used for each experiment. Figure 8 As shown, GUN4 containing two transposon insertionsINS The ability of the promoter of the genotype material to drive the expression of the reporter gene is higher than that of the promoter of the GUN4 genotype material.

Claims

1. A molecular marker for identifying waterlogging tolerance in Brassica napus, characterized in that: The sequences of the molecular markers are shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. A kit for identifying waterlogging tolerance of Brassica napus, characterized in that: The kit contains reagents for detecting the molecular markers according to claim 1.

3. The kit according to claim 2, characterized in that The primer sequences for detecting the molecular markers described in claim 1 are shown in SEQ ID NOs. 3 and 4.

4. Use of the molecular marker according to claim 1 or the kit according to claim 2 or 3 in identifying rapeseed waterlogging tolerance or in rapeseed breeding.

5. The use according to claim 4, characterized in that: The rapeseed containing the molecular marker described in claim 1 has strong waterlogging resistance.

6. A method for identifying waterlogging tolerance of Brassica napus, characterized in that: The method comprises detecting whether the rapeseed to be tested contains the molecular marker described in claim 1, and the rapeseed containing the molecular marker described in claim 1 has high waterlogging tolerance.

7. The method according to claim 6, characterized in that The molecular marker of claim 1 is amplified using primers, wherein the primer sequences are shown in SEQ ID NOs. 3 and 4. If a 4.4 Kb product is obtained by amplification, it has high waterlogging resistance, and if a 537 bp product is obtained by amplification, it has low waterlogging resistance.

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